INTRODUCTION

Invasive species are key threats to agronomic and natural ecosystems. The tomato leafminer (TLM), Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), is an invasive pest which has spread its geographic range to many parts of the world in recent years. Features such as high reproductive potential, relatively short life cycle, and the invasive feeding behavior of this insect have caused difficulties in the successful control of this pest and have facilitated the development of pesticide resistance (Batalla-Carrera et al., 2010, Barati et al., 2018, Waiba et al., 2021a).  The larvae of T. absoluta can penetrate and feed on all aerial parts of host plants such as; stems, leaves, buds, and fruits (Desneux et al., 2010). Since its recognition as an economic pest, chemical control has been the major strategy for controlling this pest (Lietti et al., 2005).In India, T. absoluta was first-time detected at the central-west (Maharashtra)(Sridhar et al., 2014) in October 2014. The new invasive pest, tomato pinworm/leaf minor, T. absoluta was recorded first in Pune in tomato plant which grown in field and poly-house in 2014 and south-west India (Karnataka state) (Anonymous, 2015, Vysali et al., 2021), and to our knowledge, there are no reports of this pest in the east and north-eastern India. Subsequently, the pest has been reported in the farmer’s fields in major tomato growing states including Himachal Pradesh.

Entomopathogenic nematodes (EPNs) from the families Heterorhabditidae and Steinernematidae are soil-inhabiting organisms that are obligate insect parasites in nature (Kaya and Gaugler, 1993, Kasi et al., 2020). These nematodes have evolved a mutualistic association with bacteria in the genera Photorhabdus is associated with Heterorhabditis spp., is carried in the intestine of infective juveniles (IJs) (Bird and Akhurst, 1983, Arthurs et al., 2004, Silva et al., 2002). Xenorhabdus is connected with Steinernema spp. and confined to a specific vesicle within the intestine of the IJs. Nematodes locate their potential host by following insect cues (Lewis et al., 2006).After IJs locate a host, they infect it through an orifice such as the mouth, anus, or spiracles or by penetrating the cuticle (particularly in Heterorhabditis spp.). Once IJs enter the host, they shed their outer cuticle(Sicard et al., 2004) and begin ingesting hemolymph, which triggers the release of symbionts by defecation (in Steinernema spp.) or regurgitation (in Heterorhabditis spp.) (Martens et al., 2004, Grewal et al., 2005, Waiba et al., 2021b). The nematode–bacteria complex kills the host within 24 - 48h through septicemia or toxemia (Dowds and Peters, 1971, Forst and Clarke, 2002). Bacteria recolonize the nematodes, which emerge as IJs from the depleted insect cadaver in search of new hosts (Poinar 1990, Kasi et al., 2021b).

More than 100 species of EPNs have been identified globally (approximately 80% are steinernematid) and at least 13 of these species have been commercialized (Shapiro-Ilan et al., 2014, Sharma et al., 2022). Commonly, the innate virulence against different pest species varies among EPN species. Moreover, differences among EPN species in host-seeking strategy and tolerance to environmental conditions like temperature and desiccation can determine the field efficacy of EPNs (Martens et al., 2004). EPNs have been broadly used in the biological control of a variety of economically important pests occupying different habitats (Grewal et al., 2005). However, EPN formulation to retard desiccation or the addition of adjuvants to increase leaf coverage and persistence of the IJs has enhanced the use of EPNs against foliar pests (Arthurs et al., 2002, Head et al., 2004, Kasi et al., 2021a, Kasi et al., 2021b     ). Our objective The evaluation of local isolated Strains of entomopathogenic nematodes, use to new Combination with Low-Toxicity Insecticides at Low and High Dosages South American Tomato Pinworm, Tuta absoluta (Meyrick) (Lepidoptera, Gelechiidae) as a biocontrol.

MATERIALS AND METHODS

The laboratories experiments were conducted during September, 2021 to test the pathogenicity of two EPNs species S. feltiae and H. bacteriophora at different (IJs cm^-2) concentrations against Tuta absoluta (Meyrick)(Lepidoptera, Gelechiidae), in Department of Entomology, Nematology Laboratory, Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India.

2.1.  Insects

T. absoluta colony was maintained on tomato plants under greenhouse conditions. The colony had been established from larvae collected in September 2021 from the vegetable science departmental form tomato greenhouse in Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Solan, HP, India, that used local strains S. feltiae (HR1) and H. bacteriophora (HR2) indigenous strains (Poinar) for the pest’s management.

2.2.  Entomopathogenic nematodes

Two isolates of S. feltiae and H. bacteriophora were used in this study. They used directly in the experiments without culturing. The native isolate was obtained from soil samples, collected from Rajgarh, Himachal Pradesh, India using G. mellonella larvae as nematode traps. This isolate was cultured based on the method(Kaya and Gaugler, 1993) at 21±1°C on the last instar larvae of G. mellonella. Infective juveniles (IJs) that emerged during the first ten days were collected from white traps stored at 4°C in distilled water for up to 14 days. The nematodes were acclimatized at room temperature for about 30 m before being used in the experiments.

2.3.  Rearing of tomato pinworm, Tuta absoluta

The larvae and pupae of TLM were collected from an infested greenhouse. T. absoluta was reared in the greenhouse, at 26±2°C, 60±10% RH, and an L:D 8:16 photoperiod. The insects were reared in wooden framed cages covered with 80 mesh organdy cloth on tomato plants (S. lycopersicum L.) (Figure 1).

Figure 1: Invasive pest of tomato pinworm Tuta absoluta rearing

The adults were fed by 10% sugar solution in the oviposition cage.

2.4.  Methodology

Ten larvae were placed in an 8 cm petri dish containing tomato leaf’s natural diet. Low and high registered label dosages (converted to lab dosages) of 2 biological agents, 3 synthetic insecticides, and the biological agent+low-toxicity insecticides were used (Table 1). 10 larvae were treated topically with 200 µl of insecticide solution dosage^-1. The control was treated only with distilled water. Treated Petri dishes were held in a CRD design with 4 replications (total n=40 larvae dosage^-1) in the lab at 18–20°C and photoperiod 12:12 h (L:D). Larval mortality was evaluated between 24 and 96 h after application.

2.5.  Statistical analysis of data

Abbott’s formula, (Abbott, 1925) was used to correct the data for control larval mortality in the bioassays and PROBIT analysis was conducted. Also, LSD (p<0.05) values were calculated to differentiate means among treatments.

Table 1: Active ingredients and laboratory dosages to evaluate the efficacy of 2 biological agent, 3 synthetic insecticides, and least toxic combinations of insecticides to control Tuta absoluta larvae

RESULTS AND DISCUSSION

3.1.  Bioassays with S. feltiae+T. absoluta+Insecticides

In general, a higher mean % mortality was noted at higher dosages (F=52.99; df=12; p<0.05) (Table 2). However, S. feltiae and Spinosad caused 80–97% of larvae mortality with low and high dosages at 96 h. The use of chlorantraniliprole and Spinosad resulted in % of mortality up to 97.50% at 96 h at the higher dosage. These results are different from those reported (Belay et al., 2012), where Spinosad and chlorantraniliprole caused larvae mortality over 80% at the same period. The LD₅₀ was 1600 ppm for chlorantraniliprole and 400 ppm for Spinosad at 96 h in this study (Figure 2).

Figure 2: Mortality of S. feltiae IJs exposed for 96 h to different concentrations of Spinosad, Chlorantraniliprole, Indoxacarb. Means with different letter are significantly different (p<0.05)

Differences might be related to (1) the prolonged use of these active ingredients caused some level of resistance, (2) instar stage, third vs fifth used in this research, or (3) differences among fall armyworm populations on the island (Viteri et al., 2018). In contrast, indoxacarb caused 72.50 and 90% of larvae mortality in low and high doses, respectively, at 96 h (Figure 5 and 6).

The use of S. feltiae in combination with Spinosad and chlorantraniliprole produced higher % of mortality at 24 h compared with the use of these insecticides alone regardless of the dosage used (Table 2). Furthermore, the highest % of larval mortality (over 95%) were noted with high dosages at 72 h. Likewise, the combination of Spinosad+S. feltiae was effective (70.00% of dead larvae) with the high dose at 96 h compared with the lowest mortality caused by Spinosad (20.50%), or S. feltiae (45.00%) applied alone (Viteri et al., 2018). Larvae exposed to 2 different modes of action [septicemia (S. feltiae)+lysed midgut epithelial cells (Spinosad), impaired regulation of muscles (chlorantraniliprole), or Indoxacarb neural transmission (spinetoram)](Viteri et al., 2018) at the same time, caused their higher mortality. In fall armyworm populations from Florida (Yu, 1991). However, further research is required to corroborate this synergistic effect (Figure 4).

Figure 3: Mortality of H. bacteriophora IJs exposed for 96 h to different concentrations of Spinosad, Chlorantraniliprole, Indoxacarb. Means with the different letters are significantly different (p<0.05)

Figure 4: Bio-efficacy of EPN’s against Tuta absoluta (M.)

Figure 5: After treatment tomato pinworm Tuta absoluta

Figure 6: After nematode infected dead tomato pinworm larvae dissected under microscopic condition and conformation Synergistic activity

Table 2: Mortality percentage of tomato pinworm [Tuta absoluta M.] larvae in the final-instar at low and high dosages of 3 insecticides, and least toxic combinations among them evaluated from 24–96 h

3.2.  Bioassays with H. bacteriophora+T. absoluta+Insecticides

In general, a higher mean % mortality was noted at higher dosages (F=72.98; df=12; p<0.05) (Table 3). However, H. bacteriophora and Spinosad caused 75.00–100% of larvae mortality with low and high dosages at 96 h. The use of chlorantraniliprole and Spinosad resulted in % of mortality up to 97.50% at 96 h at the higher dosage. These results are different from those reported (Belay et al., 2012), where Spinosad and chlorantraniliprole caused larvae mortality over 95% at the same period. The LD₅₀ was 400 ppm for Spinosad and 1600 ppm for chlorantraniliprole and at 96 h in this study (Figure 3). Differences might be related to (1) the prolonged use of these active ingredients caused some level of resistance, (2) instar stage, third vs fifth used in this research, or (3) differences among fall armyworm populations on the island (Viteri et al., 2018, Martens and Goodrich-Blair 2005). In contrast, indoxacarb caused 67.50 and 95.00% of larvae mortality in low and high doses, respectively, at 96 h (Figure 5 and 6).

Table 3: percentage of mortality of tomato pinworm [Tuta absoluta M.] larvae in the fifth-instar at low and high dosages of 3 insecticides, and least toxic combinations among them evaluated from 24–96 h

CONCLUSION

High EPN efficacy obtained under laboratory conditions cannot easily be extrapolated to field efficacy. Therefore, were field experiments on tomato crops are justified to fully determine the potential of local EPN isolates against T. absoluta in Himachal Pradesh conditions. In combination with low-toxicity insecticides at low and high dosages to control fifth-instar larvae in bioassays.

ACKNOWLEDGEMENT

This work was financed by grants from the All India Coordinated Research Project HCR-193-07 (AICRP on Nematodes). We are grateful to Nematology Laboratory, Department of Entomology, Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India-173230. And Indian council of Agricultural Research (ICAR) for granting access to laboratories facilities.

Reference

Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. Journal of Econnomic Entomology 18, 265–267.

Arthurs, S., Heinz, K.M., Prasifka, J.R., 2004. An analysis of using entomopathogenic nematodes against above-ground pests. Bulletin of Entomological Research94(4), 297–306.

Barati, R., Hejazi, M.J., Mohammadi, S.A., 2018. Insecticide susceptibility in Tuta absoluta (Lepidoptera: Gelechiidae) and metabolic characterization of resistance to diazinon. Journal of Economic Entomology111, 1551–1557.

Batalla-Carrera, L., Morton, A., Garcia-del-Pino, F., 2010. Efficacy of entomopathogenic nematodes against tomato leafminer Tuta absoluta in laboratory and greenhouse conditions. BioControl 55(4), 523–530.

Belay, D.K., Huckaba, R.M., Foster, J.E., 2012. Susceptibility of the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), at Santa Isabel, Puerto Rico, to different insecticides. Florida Entomology95, 476–478.

Bird, A.F., Akhurst, R.J., 1983. The nature of the intestinal vesicle in nematodes of the family Steinernematidae. International Journal of Parasitology13, 599–606.

Desneux, N., Wajnberg, E., Wyckhuys, K.A.G., Burgio, G., Arpaia, S., Narvaez-Vasquez, C.A., Gonzalez-Cabrera, J., Ruescas, D.C., Tabone, E., Frandon, J., Pizzol, J., Poncet, C., Cabello, T., Urbaneja, A., 2010. Biological invasion of European tomato crops by Tuta absoluta: Ecology, geographic expansion and prospects for biological control. Journal of Pest Science83(3), 197–215.

Dowds, B.C.A., Peters, A., 2002. Virulence mechanisms. In: Gaugler, R. (Ed.). Entomopathogenic Nematology. CABI Publishing, New York, USA, 79–98.

Forst, S., Clarke, D., 2002. Bacteria–nematode symbiosis. In: Gaugler, R. (Ed.). Entomopathogenic Nematology. CABI Publishing, New York, USA, 57–77.

Grewal, P.S., Ehlers, R.U., Shapiro-Ilan, D.I., 2005. Nematodes as Biocontrol Agents. CABI Publishing, Wallingford, UK.

Head, J., Lawrence, A.J., Walters, K.F.A., 2004. Efficacy of the entomopathogenic nematode, Steinernema feltiae, against Bemisia tabaci in relation to plant species. Journal of Applied Entomology 128(8), 543–547.

Kasi, I.K., Singh, M., Waiba, K.M., Monika, S., Waseem, M.A., Archie, D., Gilhotra, H., 2021a. Bio-efficacy of entomopathogenic nematodes, Steinernema feltiae and Heterorhabditis bacteriophora against the Cabbage butterfly (Pieris brassicae [L.]) under laboratory conditions. Egyptian Journal of Biological Pest Control 31, 125.

Kasi, I.K., Singh, M., Waiba, K.M., Monika., 2021b. Occurrence and distribution of entomopathogenic nematodes in soils of Solan and Sirmaur district of Himachal Pradesh, India. International Journal of Agriculture, Environment and Biotechnology 14(3), 393–397.

Kasi, I.K., Waiba, K.M., Singh, M., 2020. First report of natural infestation of Ovomermis sinensis (Nematoda: Mermithidae) parasitizing fall armyworm Spodoptera sp. (Lepidoptera: Noctuidae) in Himachal Pradesh, India. Indian Journal of Nematology 50(2), 148–149.

Kaya, H.K., Gaugler, R., 1993. Entomopathogenic nematodes. Annual Review of Entomology 38, 181–206.

Lewis, E.E., Campbell, J.C., Griffn, C., Kaya, H.K., Peters, A., 2006. Behavioral ecology of entomopathogenic nematodes. Biological Control 38, 66–79.

Lietti, M.M., Botto, E., Alzogaray, R.A., 2005. Insecticide resistance in Argentine populations of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae). Neotropical Entomology34(1), 113–119.

Martens, E.C., Goodrich-Blair, H., 2005. The Steinernema carpocapsae intestinal vesicle contains a sub-cellular structure with which Xenorhabdus nematophila associates during colonization initiation. Cellular Microbiology7(12), 1723–1735.

Martens, E.C., Vivas, E.I., Heungens, K., Cowles, C.E., Goodrich-Blair, H., 2004. Investigating mutualism between entomopathogenic bacteria and nematodes. In: Cook, R., Hunt, D.J. (Eds.). Proceedings of the Fourth International Congress on Nematology 8–13 June 2002, Nematology Monographs and Perspectives (Volume 2), 447–462.

Poinar, G.O., 1990. Biology and taxonomy of Steinernematidae and Heterorhabditidae. In: Gaugler, R., Kaya, H.K. (Eds.). Entomopathogenic Nematodes in Biological Control. CRC Press, Boca Raton, 23–58.

Shapiro-Ilan, D., Han, R., Qiu, X., 2014. Production of entomopathogenic nematodes. In: Morales-Ramos, J., Rojas, G., Shapiro-Ilan, D.I. (Eds.). Mass Production of Beneficial Organisms: Invertebrates and Entomopathogens. Academic Press, Elsevier Inc., San Diego, California, USA, 321–356.

Sharma, A., Daroch, R.K., Kapoor, R., Kasi, I.K., 2022. Status of bee keeping in Himachal Pradesh, India: A review. The Pharma Innovation Journal 11(3), 257–265.

Sicard, M., Brugirard-Ricaud, K., Pages, S., Lanois, A., Boemare, N.E., Brehelin, M., Givaudan, A., 2004. Stages of infection during the tripartite interaction between Xenorhabdus nematophila, its nematode vector, and insect hosts. Applied and Environtal Microbiology70(11), 6473–6480.

Silva, C.P., Waterfield, N.R., Daborn, P.J., Dean, P., Chilver, T., Au, C.P.Y., Sharma, S., Potter, U., Reynolds, S.E., Ffrench-Constant, R.H., 2002. Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta. Cellular Microbiology4(6), 329–339.

Sridhar, V., Chakaravarthy, A.K., Asokan, R., Vinesh, L.S., Rebijith, K.B., Vennila, S., 2014. New record of the invasive South American tomato leafminer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) in India. Pest Management in Horticultural Ecosystems 20(2), 148–154.

Viteri, D.M., Linares, A.M., Flores, L., 2018. Use of the entomopathogenic nematode Steinernema carpocapsae in combination with low-toxicity insecticides to control fall armyworm (Lepidoptera: Noctuidae) larvae. Florida Entomology101(2), 327–329.

Vysali, P., Subramanyam, K., Kasi, I.K., 2021. A study on the management of biotic and abiotic threats in chilli crop cultivation. Pharma Innovation Journal 10(12), 1741–1748.

Waiba, K.M., Chowdary, C., Khanal, B., Adhikari, B., Khadka, H., Bista, U.B., Kasi, I.K., 2021b. Effect of different organic and inorganic fertilizer on vegetative, yield and post-harvest characteristics of selected varieties of tomato (Solanum lycopersicum L.) under protected condition in Himalayan region of Nepal. International Journal of Agriculture, Environment and Biotechnology 14(03), 365–374.

Waiba, K.M., Sharma, P., Kasi, I.K., Chauhan, S., 2021a. Studies of Genetic Variability of Tomato (Solanum lycopersicum L.) Hybrids under Protected Environment. International Journal of Bio-resource and Stress Management 12, 264–270

Yu, S.J., 1991. Insecticide resistance in the fall armyworm, Spodoptera frugiperda (J. E. Smith). Pesticide Biochemistry and Physiology39(1), 84–91.